A stem cell is a cell that has the ability to replicate itself for indefinite periods and to give rise (differentiate), under suitable conditions, to the many different cell types that make up an organism. That is, stem cells have the ability to develop into mature cells that have characteristic morphology and specialized functions, such as liver cells, heart cells, skin cells, nerve cells, etc.
Embryonic stem (ES) cells are able to differentiate into virtually all cell and tissue types of an organism—in vivo as well as in vitro. Unlike other stem cells, they can differentiate into cells that are derived from all three primary germ layers: the ectoderm, mesoderm or endoderm. Each cell type and tissue type in an adult organism originates from these three primary germ layers. For example, the endoderm is the source of epithelial cells lining respiratory passages and the gastrointestinal tract and gives rise to the pharynx, esophagus, stomach and intestine and many glandular tissues and organs, including salivary glands, liver, pancreas and lungs.
The development of effective tissue-engineered artificial liver devices, generated from autogenic or allogeneic cells, has been limited by the absence of large numbers of mature, functional hepatocytes. Such artificial liver devices can be used, for example, as a true alternative to liver transplantation.
Also, devices employing functional hepatocytes can be used as in vitro systems for pharmaceutical toxicology studies, and as biosensors for environmental toxins. However, the development of these devices has similarly been limited by the absence of large numbers of mature functional hepatocytes.
Recently, considerable effort has been directed towards incorporating adult and embryonic stem cell differentiation strategies into the generation of a renewable hepatocyte cell source (1, 2, 3, 4 and 5). Although several publications have described techniques to effectively differentiate stem cells into hepatocytes (6), these prior techniques have been limited in a number of ways. Such limitations include the absence of large scale processing considerations, incomplete downstream enrichment techniques, and ineffective long term maintenance of functional hepatocytes. Thus, a culture environment that is controllable, scalable and reversible may alleviate the practical limitations of effectively differentiating stem cells into hepatocytes.
In the past, scalable, controlled culture systems have been generated with alginate poly-L-lysine (PLL) encapsulation (7, 8 and 9). The benefits of encapsulating cells in alginate are numerous. First, alginate encapsulation is reversible. For example, polymerized alginate beads can be contacted with divalent cation chelators to depolymerize the alginate. This facilitates rapid cell recovery for downstream analysis and application. Second, alginate encapsulation, through variations in the encapsulation process (e.g., alginate concentration, alginate composition, PLL concentration, bead diameter, and cell seeding density), can discretely control key culture parameters.
Prior to the present invention, it was believed that full differentiation potential of ES cells could only be achieved if the ES cells were forced to differentiate both by morphological cues (embryoid body [EB] formation), as well as by growth- and differentiation-inducing factors. It was known that microencapsulated ES cells can grow as compact colonies within alginate beads and give rise to morula-like structures and subsequently to embryoid bodies. Previous investigators focused on large-scale production of embryoid bodies in alginate microbeads, with subsequent induction of differentiation into a specific cell type (e.g., cardiomyocytes) in adhesion cultures. A disadvantage of this approach was that it was necessary to release the compact colonies of ES cells from the microbeads before differentiation in adhesion cultures could be induced. Also, it was necessary to culture the bead-released ES cell colonies in the presence of retinoic acid to induce differentiation.
Since the aim is to establish a method suitable for the large-scale differentiation of ES cells into specific cell types, differentiation within the beads is desirable. For example, this permits a specific cell type to be recovered in a small culture volume.
Before the present invention, approaches to differentiate ES cells into specific cell types within alginate beads had limited success and had practical limitations. For example, previously, differentiation of ES colonies within 1.1% alginate microbeads, which had weak mechanical resistance, could only be promoted by supplementation of the cell media with retinoic acid, and required the cumbersome formation of embryoid bodies. Also, only some of the cystic embryoid bodies within the beads appeared to differentiate into a specific cell type.
In view of the foregoing, it would be advantageous if alginate microencapsulation could be used as a tissue culture environment for the controlled differentiation of embryonic stem cells within the alginate beads. It would also be advantageous to provide a system in which the beads have good mechanical resistance, and in which differentiation within the beads is possible in absence of growth factors, and in absence of embryoid body intermediates.